The purpose of this work is to understand how C. trachomatis infection persists in humans and how persistence contributes to serious post-infection sequelae such as blindness and infertility. To accomplish this goal we have performed functional genomic studies on both reference laboratory strains and clinical isolates focusing on the genes that reside in the pathogen's plasticity zone (PZ); a small polymorphic region that contains cytotoxin genes, phospholipase D genes, and the tryptophan operon (trpRBA) that encodes the last two enzymes (Trp B and Trp A) of the tryptophan biosynthetic pathway. We found that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogen's Trp A synthase. Our functional genomic studies find that the paradigm established for reference serovars also applies to clinical isolates; specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme; implicating a functional role for the synthase in the pathogenesis of chlamydial STDs. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We believe that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma mediated eradication and thus establish persistent infections. We have shown that Chlamydiae use Trp A and B to avoid the effect of IFN-gamma on host epithelial cells, a mechanism that allows the pathogen to avoid host defense and establish persistent. To date, vaccination using conventional approaches such as targeted chlamydial recombinant proteins or DNA encoding proteins have failed in eliciting a protective anti-chlamydial immunity at the genital mucosae. Our conclusions are that the antigenic complexity of the chlamydiae, its complex life cycle, and tropism for mucosal epithelial cells constitute overwhelming challenges for the generation of a conventional vaccine. Consequently, we are currently focusing on the generation of live-attenuated vaccine strains. This is being accomplished by the clonal selection of variants that are incapable of synthesizing tryptophan synthase, a key enzyme in the ability of the pathogen to persist in epithelial cells in the presence host defense. Clones whose growth is highly sensitive to IFN-gamma in vitro and that have mutations in the trpRBA genes will be tested in in vivo in pre-clinical primate models of infection for attenuated growth and pathological properties. These studies should yield information important to the development of a highly efficacious and safe vaccine capable of preventing or controlling chlamydial caused STDs in humans.